Genome-Wide Identification, Classification, and Expression Analyses of the CsDGAT Gene Family in Cannabis sativa L. and Their Response to Cold Treatment

Hempseed is a nutrient-rich natural resource, and high levels of hempseed oil accumulate within hemp seeds, consisting primarily of different triglycerides. Members of the diacylglycerol acyltransferase (DGAT) enzyme family play critical roles in catalyzing triacylglycerol biosynthesis in plants, often governing the rate-limiting step in this process. As such, this study was designed to characterize the Cannabis sativa DGAT (CsDGAT) gene family in detail. Genomic analyses of the C. sativa revealed 10 candidate DGAT genes that were classified into four families (DGAT1, DGAT2, DGAT3, WS/DGAT) based on the features of different isoforms. Members of the CsDGAT family were found to be associated with large numbers of cis-acting promoter elements, including plant response elements, plant hormone response elements, light response elements, and stress response elements, suggesting roles for these genes in key processes such as development, environmental adaptation, and abiotic stress responses. Profiling of these genes in various tissues and varieties revealed varying spatial patterns of CsDGAT expression dynamics and differences in expression among C. sativa varieties, suggesting that the members of this gene family likely play distinct functional regulatory functions CsDGAT genes were upregulated in response to cold stress, and significant differences in the mode of regulation were observed when comparing roots and leaves, indicating that CsDGAT genes may play positive roles as regulators of cold responses in hemp while also playing distinct roles in shaping the responses of different parts of hemp seedlings to cold exposure. These data provide a robust basis for further functional studies of this gene family, supporting future efforts to screen the significance of CsDGAT candidate genes to validate their functions to improve hempseed oil composition.


Introduction
Hemp (Cannabis sativa L.) is an annual herbaceous plant of the Cannabaceae family. As one of the oldest domestic crops, hemp has been widely used due to its industrial [1], ornamental [2], nutritional [3], and pharmaceutical [4] potential. Hempseed oil is a nutrient-rich edible oil with a lipid content of 25-35% and a unique fatty acid (FA) profile characterized by high levels of polyunsaturated fatty acids (PUFAs) and low levels of saturated FAs (SFAs) [5]. Depending on environmental and genetic factors, hempseed oil can consist of up to 90% unsaturated FAs, of which more than 70% are composed of PUFAs [6]. Hempseed oil is often regarded as a source of high levels of the essential fatty acids (EFAs) linoleic acid (18:2, n-6, LA) and α-linolenic acid (18:3, n-3, ALA), which are essential for human health and can only be obtained from dietary sources [6]. Hempseed oil also contains intermediate FAs such as γ-linolenic acid (18:3, n-6, GLA) and stearidonic acid (18:4, n-3, SDA) independent of precursor FAs [7]. These long-chain PUFAs (LCPUFA) are uniquely characteristic of hempseed, an ideal resource for stearidonic acid, which can serve as a precursor for the production of long-chained n-3 PUFAs [8]. LCPUFAs are physiologically essential regulators of neuronal development, inflammation, neurodegenerative activity, and cardiovascular health, yet they cannot be obtained from other common industrial oilseed crops.
Like most plant storage oils, hempseed oil primarily accumulates within the seeds, consisting mainly of triglycerides (TAGs) synthesized within the endoplasmic reticulum (ER) prior to accumulation in the form of lipid droplets (LDs) in the cytosol [9,10]. In oil crops, the primary mechanism that governs lipid assembly is the Kennedy pathway, which utilizes acyl-CoA as a substrate for fatty acyl residues and converts glycerol 3phosphate to TAG in a four-step process [11]. The final step in this process, and the only rate-limiting step, is catalyzed by Acyl-CoA: Diacylglycerol Acyltransferase (DGAT, EC 2.3.1.20), which facilitates the esterification of a fatty acyl moiety to diacylglycerol (DAG) molecule to produce TAG, committing these molecules to the biosynthesis of TAG and shaping the carbon flow toward TAG production [12]. Therefore, diacylglycerol (DAG), the substrate of DGAT, accumulates during periods of rapid lipid formation in Brassica napus [13]. Generally, the DGAT gene family members can be divided into four subfamilies: the DGAT1, DGAT2, DGAT3, and Wax Ester Synthase (WS)/DGAT. The first DGAT gene in eukaryotes has been cloned in mice [14,15]. Members of the DGAT1 and DGAT2 subfamilies are transmembrane acyltransferases that localize to the ER with different membrane topologies and function as the primary isoenzymes to TAG synthesis in seed plants [14,15]. These two enzymes are thus likely to have evolved separately before undergoing functional convergence due to their similar acyltransferase activities utilizing DAG as a substrate [16]. Members of the DGAT3 subfamily are soluble cytosolic enzymes initially identified in peanut cotyledons [17]. Members of the dual-function WS/DGAT subfamily were initially characterized in A. thaliana and primarily catalyzed the biosynthesis of wax esters [18]. In addition, WS/DGAT enzymes catalyze the final step in producing small quantities of TAG, and their activity has been analyzed in many different plants [19].
The lipid accumulation process within plant tissues is complex and regulated by many genes [20]. Efforts to increase oilseed plant oil yields have spurred research focused on the identification and characterization of genes involved in the regulating of this agronomically valuable process so that breeders can seek to mutate or otherwise manipulate these genes to enhance oil quality [16]. Many studies of oil-rich plants, including Glycine max, Arachis hypogaea, Brassica napus, and Vernicia fordii, have emphasized the critical central role of reactions catalyzed by DGATs in the determination of the degree of TAG assembly and accumulation [21][22][23][24]. The transfer of the soybean GmDGAT1-2 gene into Arabidopsis, for example, was associated with a 1.2-fold increase in total FA levels in the resultant transgenic plants relative to wild-type controls [25]. Overexpressing the peanut-derived AhDGAT2a gene in tobacco seeds impacted the transcription level of endogenous tobacco lipid metabolism genes and enhanced the FA content therein [26]. When the BnDGAT genes were overexpressed, this was sufficient to restore the synthesis of TAG in yeast H1246 while promoting FA accumulation therein [27].
Additionally, diverse environmental factors can impact plant oil formation, such that lipid-associated genes can govern blot growth and stress responses [28,29]. Plant lipid metabolic intermediates are important components of biofilm and active signal molecules, which are easily affected by environmental factors [30]. Low temperature is the leading environmental factor affecting crops' geographical distribution, yield, and quality, which is closely related to fatty acids, protein, and other nutrients of hempseeds. Previous studies have shown that plants can respond to cold stress by accumulating lipids such as phosphatidic acid (PA), diacylglycerol (DAG), and triglyceride (TAG) in vivo [30]. DGAT is a vital substrate competitor for phospholipid and glycolipid synthesis, an important component of biofilm, so its coding genes play a crucial role in coordinating lipid metabolism [15].
It was shown that the conversion of phosphatidylcholine (PC) to triacylglycerol (TAG) was higher under cold treatment, and the expression of the DGAT1 gene in cold-tolerant plants is higher than that in sensitive plants during a cold response, which promotes the accumulation of TAG in response to cold stress [31]. A. thaliana transforms DAG into TAG by overexpressing the AtDGAT2 gene under cold stress to improve the plant's cold tolerance [28]. The study on the cold response mode of the DGAT gene in hemp is of great value in improving the cold tolerance of hemp. However, it is unclear how many DGAT genes are contained in hemp, the function of its members, and the cold response pattern.
Hemp has long been cultivated and is a natural source of nutrients essential for optimal nutrition [32]. While DGAT enzymes are known to control the rate-limiting step in the process of lipid synthesis, the specifics of the copy numbers, sequences, structure, evolutionary relationships, and spatiotemporal expression patterns of CsDGAT genes (CsDGATs) have yet to be analyzed comprehensively. Accordingly, the present study was constructed as a comprehensive bioinformatics analysis of the CsDGAT gene family. To aid efforts to improve the yield and quality of hempseed oil, we herein analyze the genomic architecture for different isoforms of the four classes of hemp DGAT genes (DGAT1, DGAT2, DGAT3, WS/DGAT) in comparison to those of other plant species. In addition, the gene structures, conserved domains, transmembrane domains, chromosomal positioning and collinearity, phylogenetic associations, subcellular localization, and cis-acting elements associated with these CsDGATs were characterized. Moreover, the expression of the identified CsDGATs in different tissues and response to cold stress conditions was evaluated using qPCR and transcriptomic approaches. Together, these findings offer new insight regarding the evolution of the CsDGAT gene family and the properties of these genes, thereby providing a basis for further studies of their biological functions in the context of C. sativa growth and development.

Identification of C. sativa DGAT Gene Family Members
DGAT family proteins have been identified in various plant species [33]. To systematically identify candidate DGAT genes within the C. sativa genome, a hidden Markov model (HMM) and BLASTP methods were used together with whole-genome scanning. Following Pfam and Smart verification, redundant sequences in the annotated C. sativa genome were removed. The remaining 10 putative CsDGAT genes identified via this approach were tentatively designated CsDGAT1, CsDGAT2, CsDGAT3, CsWSD1.1, CsWSD1.2, CsWSD1.3, CsWSD1.4, CsWSD1.5, CsWSD1.6, and CsWSD1.7 based on their subfamilies. The proteins encoded by these genes exhibited an average length of 480 amino acids, ranging from 327 aa (LOC115703123) to 556 aa (LOC115708250). The predicted molecular weights (MWs) of these proteins ranged from 37-62 kDa, while their predicted isoelectric point (pI) values ranged from 7.0-9.5, indicating that these proteins are likely to be alkaline (Table 1). Two online tools were used to predict the subcellular localization of the encoded proteins, suggesting that CsDGAT1 and CsDGAT2 would uniformly localize to the ER membrane or plasma membrane. In contrast, CsDGAT3 and different WS/DGAT family proteins were predicted to localize to diverse cellular compartments.

Phylogenetic Analyses of CsDGAT Gene Family Members
To understand the evolutionary relationships between CsDGAT genes and those of related plant species, a neighbor-joining (NJ) phylogenetic tree incorporating the protein sequences for 162 DGAT genes across 50 representative eukaryotic species, including eudicots, monocotyledons, ferns, and algae was constructed using MEGA X (Table S1), with results being confirmed using a maximum likelihood (ML) tree. DGAT amino acid sequences yielded a well-resolved tree in which the four major DGAT subfamilies were separated from one another, including DGAT1, DGAT2, DGAT3, and WSD clades (Figure 1). Monocots and eudicots also form distinct clusters within these clades. Consistent with the tentative assignments shown above, CsDGAT1, CsDGAT2, CsDGAT3, and CsWSD proteins respectively clustered with the DGAT1, DGAT2, DGAT3, and WSD clades. The CsDGATs identified herein were more closely related to eudicot DGAT enzymes and were separated from those encoded by other species included in this analysis. These data suggest that members of the CsDGAT gene family may have the same function as collinear genes.  Table S1.  Table S1.

Chromosomal Distribution and Collinearity Analyses of CsDGAT Gene Family Members
The NCBI database was used to guide the mapping of these putative CsDGAT genes to the C. sativa genome, revealing their uneven distribution across five hemp chromosomes, with 1-5 genes per chromosome. Chromosome 1 encoded five of these CsDGAT genes (Figure 2A), while chromosomes 2, 5, and 6 only encoded a single CsDGAT, and the X chromosome encoded two CsDGATs. Notably, the number of CsDGAT genes per chromosome was not solely associated with chromosome size, given that the largest chromosome (Chr X) only encoded two CsDGATs. These CsDGAT genes were spread roughly equally across chromosomes 2, 5, and 9. These findings demonstrate that CsDGAT family genes were not evenly distributed across the five C. sativa chromosomes.

Analyses of CsDGAT Protein Structures and Conserved Domains
The genomic and protein-coding sequences of these CsDGATs were next utilized to conduct intron/exon structural analyses using the GSDS web tool. These analyses revealed distinct intron/exon structures for the members of each of these DGAT subfamilies ( Figure  3A  To more fully explore the evolution of these homologous CsDGATs, comparative syntenic maps were generated by comparing hemp to other representative species, including nine dicots (Glycine max, Brassica napus, Arachis hypogaea, Arabidopsis thaliana, Ricinus communis, Sesamum indicum, Helianthus annuus, Gossypium darwinii, Juglans regia) and three monocots (Zea mays, Oryza sativa, and Sorghum bicolor; Figure 2B). No duplicate pairs of genes resulting from tandem or segmental duplication were observed among these 10 CsDGAT genes. Overall, 19 DGATs exhibited collinearity relationships with identified CsDGATs, including three genes in A. hypogaea, three in G. max, three in H. annuus, four in J. regia, four in R. communis, and two in S. indicum. No such relationships were detected when comparing hemp sequences and those from the three representative monocots, consistent with a closer phylogenetic relationship with dicots than monocots ( Figure 2B). These data suggest that members of the CsDGAT gene family may have the same function as collinear genes.

Analyses of CsDGAT Protein Structures and Conserved Domains
The genomic and protein-coding sequences of these CsDGATs were next utilized to conduct intron/exon structural analyses using the GSDS web tool. These analyses revealed distinct intron/exon structures for the members of each of these DGAT subfamilies ( Figure  Proteins with highly conserved amino acid sequences, particularly in functional regions, often exhibit similar biological functions. Accordingly, the MEME software tool was employed to analyze the amino acid sequence domains of these putative CsDGATs, with five additional plant species also having been retrieved and the maximum number of motifs per protein sequence set to six. Orthologous sequences were identified among these six analyzed plant species, including six DGAT1 sequences, six DGAT2 sequences, six DGAT3 sequences, and 12 WS/DGAT sequences (Supplementary S1 file). Conserved motifs were then analyzed in these putative DGAT proteins, revealing the presence of shared protein motifs across the most highly conserved species ( Figure S1).
The NCBI Conserved Domain Database and DNAMAN software were additionally used to characterize the structures of these DGAT proteins. This approach revealed that Cs-DGAT1 harbored a C-terminal MBOAT (PF03062) domain between amino acids 177 and 533 and nine conserved transmembrane regions that reaffirm its identity as a membrane-bound O-acyl transferase ( Figure 3B). Multiple sequence alignment additionally demonstrated the presence of an Acyl-CoA binding signature (R 135 -G 153 ), a putative catalytic active site (R 168 LIIEN 173 ), a phosphopantetheine attachment site (G 177 to M 199 ), an SnRK1 target site (F 217 -X-X-X-X-X-I 223 -X-X-X-VV 228 ), a putative thiolase acyl-enzyme intermediate signature (C 242 -P-XX-V-X-L-R-X-DSA-X-LSG-XX-L-XXX-A 264 ), a putative fatty acid protein signature (A 408 E 409 -X-L-X-FGDREFY-X-DWWN 424 ), a DAG-binding motif (HRW-XX-RH-X-Y-X-P) and a putative C-terminal ER retrieval motif (-YYHDV-) domain in this protein (Supplementary Figure S2).
CsDGAT2 was found to harbor a PlsC domain located between amino acids 124 and 232, classifying it as a lysophospholipid acyltransferases (LPLAT) superfamily member (PF03982), with two N-terminal transmembrane domains also being identified in this analysis ( Figure 3B). Alignment analyses conducted for 70 species of plants, animals, and microbes revealed six highly conserved domains in members of the DGAT gene family [34]. Here, CsDGAT2 sequence alignment with the DGAT2 proteins encoded by Arabidopsis (AT3G51520), peanut (AEO11788), and rice (Os02g48350), soybean (Glyma01G156000), and maize (GRMZM2G050641) was performed, revealing conserved PH Block, PR Block, GGE Block, RGFA Block, VPFG Block, and G Block motifs, in line with prior research evidence ( Figure S2).
CsDGAT3 was not found to encode any transmembrane domains and contained a single TRX domain ( Figure 3B). The comparison of the full CsDGAT3 sequence with sequences of other known acyltransferases confirmed that a phosphopantetheine attachment site was present in CsDGAT3 at S 24 -G 38 , with a putative thiolase acyl-enzyme intermediate signature S 166 XXXXXXSXXS 176 also being detected therein ( Figure S2). CsDGAT3 also har-bored a fatty acid protein binding signature (KSGSAALVEEFERVMGAE), and a putative active catalytic site was detected between the NLFRDE residues.

CsDGAT Secondary and Tertiary Structural Analyses
The structures of these 10 CsDGAT proteins were compared in greater detail, with SOPMA being used to examine their predicted secondary structural characteristics. These CsDGATs included α-helix, extended strand, beta-turn, and random coil elements (Table  S2), with over 70% of the secondary structure comprising α-helix and random coil elements. In contrast, the proportion of extended chain and beta-turn elements in the overall CsDGAT protein sequences was relatively low ( Figure S3).
A homology modeling method and the SWISS-MODEL database were further used to construct the predicted 3D structures of these CsDGAT proteins, with the optimal structure being that with the highest score. Highly significant differences were observed when comparing the 3D structures of members of these different CsDGAT subfamilies ( Figure  S4), with CsDGAT1 and CsWSD1 subfamily members being the most structurally complex. The diverse structural characteristics of these CsDGATs provide a valuable foundation for further research to clarify their physiological functions. All CsWSD proteins were found to harbor conserved N-terminal WES (PF03007) domains and conserved C-terminal DFU (PF06974) domains ( Figure 3B). Some of these CsWSD proteins contain transmembrane regions and other conserved domains. CsWSD1.2, CsWSD1.6, and CsWSD1.7, for example, each contained 1-2 transmembrane regions, while CsWSD1.3 harbors an AAtase domain (PF07247). Different members of these CsDGAT gene subfamilies may have thus evolved distinct functions. Multiple sequence alignment was also proposed for a proposed N-terminal active-site motif (HHXXXDG), revealing its presence in these candidate WS/DGAT1 coding sequences ( Figure S2).

CsDGAT Secondary and Tertiary Structural Analyses
The structures of these 10 CsDGAT proteins were compared in greater detail, with SOPMA being used to examine their predicted secondary structural characteristics. These CsDGATs included α-helix, extended strand, beta-turn, and random coil elements (Table S2), with over 70% of the secondary structure comprising α-helix and random coil elements. In contrast, the proportion of extended chain and beta-turn elements in the overall CsDGAT protein sequences was relatively low ( Figure S3).
A homology modeling method and the SWISS-MODEL database were further used to construct the predicted 3D structures of these CsDGAT proteins, with the optimal structure being that with the highest score. Highly significant differences were observed when comparing the 3D structures of members of these different CsDGAT subfamilies ( Figure S4), with CsDGAT1 and CsWSD1 subfamily members being the most structurally complex. The diverse structural characteristics of these CsDGATs provide a valuable foundation for further research to clarify their physiological functions.

Identification of CsDGAT Promoter cis-Acting Elements
Cis-acting regulatory elements are short sequences within the promoter regions upstream of particular genes that can be recognized and bound by specific transcription factors, thereby regulating gene expression. The 2000 bp promoter regions upstream of each CsDGAT gene were next analyzed to identify predicted cis-acting elements. This approach led to the identification of 46 such elements related to stress responses, light responses, phytohormone regulation, and plant development ( Figure 4). The CsDGAT promoter regions contained many lights and phytohormone response elements, including 11 G-box elements and seven ABRE elements in the CsDGAT3 promoter. These analyzed promoters also detected many other stress-response elements were also detected in these analyzed promoters, including cold-responsive, defense, and stress-responsive cis-acting elements. These findings emphasize the potential involvement of different CsDGAT genes in hemp plant development, environmental adaptation, and abiotic stress responses.

Identification of CsDGAT Promoter cis-Acting Elements
Cis-acting regulatory elements are short sequences within the promoter regions upstream of particular genes that can be recognized and bound by specific transcription factors, thereby regulating gene expression. The 2000 bp promoter regions upstream of each CsDGAT gene were next analyzed to identify predicted cis-acting elements. This approach led to the identification of 46 such elements related to stress responses, light responses, phytohormone regulation, and plant development ( Figure 4). The CsDGAT promoter regions contained many lights and phytohormone response elements, including 11 G-box elements and seven ABRE elements in the CsDGAT3 promoter. These analyzed promoters also detected many other stress-response elements were also detected in these analyzed promoters, including cold-responsive, defense, and stress-responsive cis-acting elements. These findings emphasize the potential involvement of different CsDGAT genes in hemp plant development, environmental adaptation, and abiotic stress responses. The total count of these four categories is displayed in the bar plot.

CsDGAT Subcellular Localization Analyses
Full-length CsDGAT sequences for six representative genes (CsDGAT1, CsDGAT2, CsDGAT3, CsWSD1.1, and CsWSD1.4) were fused to a GFP reporter gene under the control of the 35S promoter and introduced into N. benthamiana leaves to validate these predicted localization results. High levels of GFP expression were observed in the cytosol,

CsDGAT Subcellular Localization Analyses
Full-length CsDGAT sequences for six representative genes (CsDGAT1, CsDGAT2, CsDGAT3, CsWSD1.1, and CsWSD1.4) were fused to a GFP reporter gene under the control of the 35S promoter and introduced into N. benthamiana leaves to validate these predicted localization results. High levels of GFP expression were observed in the cytosol, membrane, and nuclei of the cells of these tobacco leaves. Specifically, the CsDGAT1-GFP, CsDGAT2-GFP, and CsWSD1.1-GFP fusion proteins localized to the ER, while CsDGAT3-GFP localized to the chloroplast compartment, and CsWSD1.4-GFP was present in the nuclei and cytosol, largely consistent with the above predictions ( Figure 5).

CsDGAT Gene Expression Patterns in C. sativa
To explore patterns of CsDGAT expression in C. sativa, RNA-seq data corresponding to female inflorescences from nine varieties of hemp and five different tissues from the Longdama 6 hemp variety (roots, stems, leaves, seeds, flowers) were used to construct gene expression heat maps and cluster diagrams ( Figure 6, Supplementary Tables S4-S6). The CsDGAT1, CsDGAT2, CsDGAT3, CsWSD1.2, and CsWSD1.7 expression varied markedly across these hemp varieties, with CsDGAT1, CsDGAT3, and CsWSD1.2 being expressed at relatively high levels. However, the other analyzed CsDGATs were largely undetectable in these different female inflorescences ( Figure 6A). CsDGATs were also expressed in tissue-specific patterns in a given hemp variety, with all 10 genes being expressed in at least one of the analyzed tissue types ( Figure 6B). While CsDGAT1, CsDGAT2, and CsWSD1.2 were expressed at high levels in seed and flower tissue samples, CsWSD1.1 and CsWSD1.7 were specifically expressed in the stem, leaf, and root samples, and CsWSD1.3 was expressed at a higher level in leaves and stems. The expression of CsWSD1.4 was only detected in seed tissue samples. CsDGAT3 expression levels tended to be high across various tissues, suggesting a role for this gene in the process of tissue development. CsWSD1.5 and CsWSD1.6 were expressed at relatively low levels in all analyzed tissues, suggesting that these genes may not be functional or that they exhibit spatiotemporally-specific expression patterns, which are unlikely to be important regulators of hemp development. Accordingly, different CsDGATs are likely to play distinct and varied roles in the growth and development of hemp plants. In addition, the analysis of the

CsDGAT Gene Expression Patterns in C. sativa
To explore patterns of CsDGAT expression in C. sativa, RNA-seq data corresponding to female inflorescences from nine varieties of hemp and five different tissues from the Longdama 6 hemp variety (roots, stems, leaves, seeds, flowers) were used to construct gene expression heat maps and cluster diagrams ( Figure 6, Supplementary Tables S4-S6). The CsDGAT1, CsDGAT2, CsDGAT3, CsWSD1.2, and CsWSD1.7 expression varied markedly across these hemp varieties, with CsDGAT1, CsDGAT3, and CsWSD1.2 being expressed at relatively high levels. However, the other analyzed CsDGATs were largely undetectable in these different female inflorescences ( Figure 6A). CsDGATs were also expressed in tissuespecific patterns in a given hemp variety, with all 10 genes being expressed in at least one of the analyzed tissue types ( Figure 6B). While CsDGAT1, CsDGAT2, and CsWSD1.2 were expressed at high levels in seed and flower tissue samples, CsWSD1.1 and CsWSD1.7 were specifically expressed in the stem, leaf, and root samples, and CsWSD1.3 was expressed at a higher level in leaves and stems. The expression of CsWSD1.4 was only detected in seed tissue samples. CsDGAT3 expression levels tended to be high across various tissues, suggesting a role for this gene in the process of tissue development. CsWSD1.5 and CsWSD1.6 were expressed at relatively low levels in all analyzed tissues, suggesting that these genes may not be functional or that they exhibit spatiotemporally-specific expression patterns, which are unlikely to be important regulators of hemp development. Accordingly, different CsDGATs are likely to play distinct and varied roles in the growth and development of hemp plants. In addition, the analysis of the expression patterns of CsDGAT genes in different seed development stages showed that the expression of CsDGAT3 and CsWSD1.4 genes were higher in the whole seed development stage, which was significantly higher than that of other genes, on the contrary, the expression levels of CsWSD1.3, CsWSD1.5, and CsWSD1.6 were lower than those of other genes. CsDGAT1 and CsDGAT3 were higher in the early and later stages of seed development, and CsWSD1.2 and CsWSD1.7 were higher in the middle stage of seed development. The expression of CsWSD1.4 is relatively high in the middle and later stages of seed development. It is worth noting that the expression of CsDGAT2 maintains a relatively stable expression level in the whole stage of seed development, indicating that CsDGAT family genes function in different stages of seed development. It also predicts that in higher plants the process of lipid accumulation process is finely and strictly regulated.  The expression patterns of these 10 CsDGAT genes were validated in different tissues via qPCR (Figure 7). This approach revealed patterns of CsDGAT expression in different tissues of the Longdama 6 hemp variety that differed somewhat from the above The expression patterns of these 10 CsDGAT genes were validated in different tissues via qPCR (Figure 7). This approach revealed patterns of CsDGAT expression in different tissues of the Longdama 6 hemp variety that differed somewhat from the above transcriptomic data trends. However, the expression of these genes tended to be higher in seeds and female flowers relative to other tissues. Relatively high levels of CsDGAT3, CsWSD1.3, CsWSD1.6, and CsWSD1.7 expression were observed in leaves, while marked differences in CsWSD1.4 expression were observed across tissue such that it was only expressed at high levels in seeds. transcriptomic data trends. However, the expression of these genes tended to be higher in seeds and female flowers relative to other tissues. Relatively high levels of CsDGAT3, CsWSD1.3, CsWSD1.6, and CsWSD1.7 expression were observed in leaves, while marked differences in CsWSD1.4 expression were observed across tissue such that it was only expressed at high levels in seeds. The Y-axis indicates the relative expression level of different genes. Three replicates were performed, and the vertical bar is the standard error. The actin7 gene was used as an internal reference. Significant differences between the different group are indicated by different letters a-d (p < 0.05).

Phenotype and CsDGAT Expression Pattern Analysis under Cold Stress Response in C. sativa
Under cold stress, the leaves of hemp plants showed symptoms of wilting and water loss, and the degree of gradual wilting deepened with the duration of stress ( Figure 8A). The morphology of the plant without cold stress was normal, and the degree of leaf wilting was not evident under short-term cold stress for 12 h; the degree of leaf wilting began to be obvious at 24 h, and the leaves of hemp plant wilted at 48 h. Severe wilting was observed after 72 h of cold stress ( Figure 8A(a)). From the leaf morphology at different treatment times, it can be seen that the degree of curling of hemp leaves increased with the extension of treatment time, indicating that cold stress led to changes in plant morphology ( Figure 8A(b)). CsDGAT gene expression patterns were next examined in the roots and leaves of C. sativa seedlings exposed to cold stress. Under these cold conditions, CsDGAT1, CsDGAT2, and CsDGAT3 genes were significantly (>2-fold) upregulated in the leaves of these seedlings in the middle and late stage of processing relative to untreated plants ( Figure 8B,C). In contrast, all CsWSDs other than CsWSD1.2, CsWSD1.4, and CsWSD1.5 were slightly downregulated relative to control conditions. The CsDGAT expression patterns observed in C. sativa roots were distinct from those observed in leaves, with all of these genes other than CsWSD1.7 being upregulated in response to cold stress at different time points. Specifically, CsDGAT1, CsDGAT2, and CsDGAT3 were significantly upregulated in the middle and late stages of processing, with consistent patterns in both root and leaf tissues relative to control samples. In contrast, CsWSD1.1, CsWSD1.4,   Figure 7. Expression patterns of the CsDGAT genes in different tissues detected by RT-qPCR. The characters on the X-axis show the different tissues of C. sativa (root, stem, leaf, flower, and seed). The Y-axis indicates the relative expression level of different genes. Three replicates were performed, and the vertical bar is the standard error. The actin7 gene was used as an internal reference. Significant differences between the different group are indicated by different letters a-d (p < 0.05).

Phenotype and CsDGAT Expression Pattern Analysis under Cold Stress Response in C. sativa
Under cold stress, the leaves of hemp plants showed symptoms of wilting and water loss, and the degree of gradual wilting deepened with the duration of stress ( Figure 8A). The morphology of the plant without cold stress was normal, and the degree of leaf wilting was not evident under short-term cold stress for 12 h; the degree of leaf wilting began to be obvious at 24 h, and the leaves of hemp plant wilted at 48 h. Severe wilting was observed after 72 h of cold stress ( Figure 8A(a)). From the leaf morphology at different treatment times, it can be seen that the degree of curling of hemp leaves increased with the extension of treatment time, indicating that cold stress led to changes in plant morphology ( Figure 8A(b)). CsDGAT gene expression patterns were next examined in the roots and leaves of C. sativa seedlings exposed to cold stress. Under these cold conditions, CsDGAT1, CsDGAT2, and CsDGAT3 genes were significantly (>2-fold) upregulated in the leaves of these seedlings in the middle and late stage of processing relative to untreated plants ( Figure 8B,C). In contrast, all CsWSDs other than CsWSD1.2, CsWSD1.4, and CsWSD1.5 were slightly downregulated relative to control conditions. The CsDGAT expression patterns observed in C. sativa roots were distinct from those observed in leaves, with all of these genes other than CsWSD1.7 being upregulated in response to cold stress at different time points. Specifically, CsDGAT1, CsDGAT2, and CsDGAT3 were significantly upregulated in the middle and late stages of processing, with consistent patterns in both root and leaf tissues relative to control samples. In contrast, CsWSD1.1, CsWSD1.4, CsWSD1.5, and CsWSD1.6 were initially downregulated and then upregulated under cold stress conditions, suggesting that the biological clock of these hemp plants may at least partially control the expression of certain CsDGATs.  The Y-axis indicates the relative expression level of different genes. Three replicates were performed, and the vertical bar is the standard error. The actin7 gene was used as an internal reference. Significant differences between the control group and cold treatment samples are indicated by * (p < 0.05) and ** (p < 0.01).

Discussion
Members of the DGAT gene family were initially characterized over two decades ago, and the enzymes encoded by these genes play a central role in vegetable oil production, metabolic regulation, and stress response leading to their widespread study in a range of plant species. They are also the only rate-limiting enzymes involved in the Kennedy pathway that governs de novo TAG biosynthesis [15,29]. In contrast to the DGAT gene families of the primary oilseed plant species, however, the hemp DGAT family remains to be characterized in detail. A growing number of studies have shown that DGAT genes are important targets for efforts to understand better the mechanisms that govern lipid metabolism and regulation [35]. The publication of the C. sativa genome has provided an opportunity to systematically characterize the key functional genes encoded therein to guide molecular breeding efforts [36]. Thus, there is a clear need to more fully explore CsDGAT gene resources to understand and regulate TAG biosynthesis in this economically important species and to provide a theoretical basis for elucidating the molecular mechanism of the key CsDGAT gene of lipid metabolism regulation pathway in the cold response of industrial hemp.

CsDGAT Gene Family Identification
This study identified 10 putative DGAT genes in the C. sativa genome, including one DGAT1, one DGAT2, one DGAT3, and seven WSD1 genes. Exon-intron structural diversification analyses are critical to developing a detailed understanding of the evolution of particular gene families across species [37]. In this study, the divergent exon-intron organization was observed among the four CsDGAT gene families, consistently exhibiting a unique evolutionary history and emphasizing the diversity of plant DGATs [14,37]. Marked differences in the length and distribution of intronic and UTR sequences were a major contributor to observed gene structure diversification [14]. Generally, only limited amino acid sequence similarity was observed between CsDGAT genes in these four different subfamilies, suggesting that they play distinct and nonredundant roles in regulating hemp physiology.
The functional domains of CsDGAT gene family members were additionally characterized, revealing conserved MBOAT (PF03062), LPLAT (PF03982), WES (PF03007), and DFU (PF06974) domains. Conserved sequences and active catalytic sites in these DGAT homologs were also analyzed, revealing that these proteins shared very low levels of identity with members of other CsDGAT subfamilies ( Figure S5). Gene synteny analyses indicated an absence of synteny among the four CsDGAT subfamilies (Figure 2), and significant differences in intron/exon gene structure were observed among these four subfamilies ( Figure 3A). Conserved domains and motifs were detected in members of all four analyzed DGAT subfamilies (Figures 3B and S2), in line with prior reports studying peanut [22] and oil palm plants [16]. While some variations in sequence homology were noted among different DGAT genes encoded by the same species, the conserved domains and functional motifs are likely to be associated with active sites that are important for substrate binding, catalytic activity, and other key regulatory roles [16].
There has been a growing research focus on the role of cis-acting elements that influence gene expression in recent years, with many of these elements being responsive to environmental stressors and phases of plant development [38]. A promoter analysis for the CsDGATs identified herein revealed several stress-responsive, light-responsive, phytohormone-responsive, and plant development-related cis-acting elements, emphasizing the potential interactions between these hormone-responsive genes and a range of other metabolic pathways. Throughout evolution, plants have developed a range of unique approaches to adapting or responding to external environmental factors. DGAT family members have been documented as central mediators of responses to biotic and abiotic stressors, including saline-alkali, cold, and drought stress conditions [29].

Phylogenetic Relationships among DGAT Family Proteins
Phylogenetic and syntenic analyses of DGATs encoded by different species have provided detailed insight into their evolution and function [14,16,29]. Members of the DGAT1 and DGAT3 gene families have convincingly been shown as originating from different ancestors during the emergence of eukaryotic species and to have evolved in a non-symmetric manner through convergent evolution [14,24]. DGAT3 and WS/DGAT family genes also form a monophyletic subfamily that is phylogenetically divergent from the DGAT1 and DGAT2 subfamily [14], with these genes being primarily encoded by plants and largely absent in animal species [14,24]. In contrast, DGAT1 and DGAT2 are widely expressed in plants, animals, and other major eukaryotic species. The specific patterns of genes in the DGAT1, DGAT2, DGAT3, and WS/DGAT subfamilies also differ from one another. In hemp, the WS/DGAT gene was the most diversified, with seven CsWSDs having been identified in the present analysis; in contrast, CsDGAT1, CsDGAT2, and CsDGAT3 were maintained as single copies, while multiple genes have been reported in the DGAT1 and DGAT2 subfamilies in many plants [14,16]. CsDGATs were most closely related to DGATs encoded by eudicots in phylogenetic analyses, indicating that they may have diverged early during the process of plant evolution, originating prior to plant diversification with DGAT activity having remained largely unchanged owing to the importance of TAGs in all species [14].

Profiling of CsDGAT Expression across Hemp Tissues and Varieties
Gene expression patterns are closely tied to the functional roles of these genes in plants [39]. DGAT enzymes are generally considered to play essential roles in the process of TAG biosynthesis [40], underscoring the value of studying these genes given the universal importance of TAGs across the various domains of life. Different DGAT genes have been shown to exhibit varying expression profiles across plant species, consistent with their species-specific functional roles [23]. Evaluating the expression profile for a particular gene across tissues can offer a foundation for subsequent research [16]. Accordingly, the expression of the putative hemp CsDGAT genes identified herein were compared through in silico and qPCR approaches, revealing gene-specific expression profiles. CsDGAT1, CsDGAT2, CsDGAT3, CsWSD1.2, and CsWSD1.7 were expressed at high levels in female inflorescences from various hemp varieties, and they may play a key role in floral development. AtDAGT1 and AhDAGT1 have previously been reported to be expressed at the highest levels in flower and seed tissues. Tissue-specific CsDGAT expression patterns were also observed within a given hemp variety, with all CsDGATs being expressed in at least one tissue compartment. For example, CsDGAT1, CsDGAT2, and CsWSD1.2 were expressed at high levels in seed and flower tissues, consistent with their potential functional roles in seed or flower development, while CsDGAT3 was highly expressed in many tissues suggesting it is more important in the context of general tissue development. CsWSD1.5 and CsWSD1.6 were expressed at relatively low levels across analyzed tissues, suggesting that they may not play major roles in the growth or development of the analyzed hemp variety.
Different patterns of DGAT gene expression were also observed across species. For example, the expression of DGAT3 in Arabidopsis is evident at high levels across different stages of development, with >2-fold upregulation evident during early seed development [41]. In contrast, peanut DGAT3 was expressed at the lowest levels in seeds and the highest in leaves and flowers [22]. CsDGAT gene expression also varies across hemp varieties, highlighting probable roles for these genes as regulators of gene expression and key functional processes. Further study of the distinct spatial expression patterns of members of the same classes of DGAT genes in different plants is warranted. Generally speaking, in oilseeds rich in normal fatty acids, such as Arabidopsis, soybean, and rapeseed, DGAT1 shows much higher expression levels than DGAT2 in developing seeds, and therefore, DGAT1 is the main enzyme for TAG synthesis in these oilseed plants [42]. However, DGAT2 is expressed at higher levels during seed development in some plant species capable of accumulating unusual fatty acids, such as Vernicia fordii [43], Cyperus esculentus [44], and Ricinus communis [45], which have a higher affinity for unusual fatty acid -containing substrates. The expression of the CsDGAT2 gene was observed to be higher than CsDGAT1 in this study, and it is noteworthy that the expression of the CsDGAT3 gene was higher in female flowers of different species, in different tissue parts and in different developmental periods of seeds, which may be associated with the formation of some specific fatty acids among hempseeds. However, the relative contribution and potential regulatory mechanism of CsDGAT genes of different family members to TAG synthesis need to be determined by more biochemical and molecular biological analysis.

The Response of CsDGAT Genes to Cold Stress
Cold stress is one of the primary environmental factors that can adversely impact the quality and yield of specific crops. Plants engage in a series of physiological and biochemical processes to adapt to cold and other abiotic stressors. Structural changes in plant cells are one of the important mechanisms by which the cell membrane system suffers first damage when plants are subjected to cold stress. It has been reported that membrane lipid composition and unsaturation are closely related to plant cold tolerance [46]. Triacylglycerol (TAG) is the main component of plant oil and an important donor source of cell membrane lipid synthesis in plants [47]. As a key enzyme for TAG synthesis, many studies have supported a potential role for CsDGAT family members in plants' cold responses [29]. Arabidopsis DGAT1 gene was induced by low-temperature stress to upregulate expression [28], and the DGAT2 gene can be induced by cold, heat, and some biotic stresses in soybean and peanut [22]. Additionally, following exposure to cold, the galactolipid MGDG undergoes conversion into DAG and oligogalactolipids, potentially further catalyzing TAG production in a manner conducive to membrane stability [29]. In this study, all 10 identified CsDGAT genes were significantly responsive to cold stress, tending to be upregulated at different time points while exhibiting distinct spatial regulatory patterns when comparing leaves and roots that suggest a possible role for CsDGAT genes as positive regulators of cold responses in hemp. Under cold stress, maize leaves were previously shown to exhibit the upregulation of 5 ZmDGAT genes, with such upregulation occurring later following cold treatment and more strongly than similar ZmDGAT gene induction observed in maize roots [29]. Root CsWSD gene responses, in contrast, were stronger than those in leaves. As such, these genes are likely to play distinct roles in different parts of hemp seedlings when responding to cold stress. In this study, the role and regulatory mode of CsDGAT gene family members in cold tolerance of hemp were initially investigated, which laid the foundation for the subsequent study of the function of the CsDGAT gene family and the selection of new cold-tolerant hemp varieties using molecular breeding methods.
In order to analyze the function of the CsDGAT gene more comprehensively, our team will construct the expression vector of the target gene promoter fused with the GUS tag for Arabidopsis transformation and analyze the expression pattern of the target gene by tissue staining method. To further validate the cold tolerance function of the CsDGAT gene, our team will continue to construct yeast expression vectors and verify the enzymatic and cold response function of the target gene in yeast lipid synthesis mutant H1246. Furthermore, the target gene will be over-expressed in Arabidopsis mutant AS11 and wild type, and the comprehensive methods of biochemistry, bioinformatics, molecular biology, and multigroup association analysis will be used to validate the lipid synthesis and cold reaction function of the target gene in plant models. The molecular mechanisms and signaling pathways involved in the cold response of the CsDGAT gene in hemp will be explained more comprehensively.

CsDGAT Gene Identification
Hemp genome sequence information was downloaded from the NCBI database (Gen-Bank assembly accession: GCA_900626175.2). Arabidopsis DGAT genes were utilized as a reference for the identification of orthologous CsDGAT gene family members. To identify other CsDGATs, particularly singleton genes, a hidden Markov model (HMM) profile was constructed from orthologous amino acid sequences and used as an HMM search query against hemp gene models using the HMMER3 package [48,49]. To identify additional hemp DGAT genes, BLASTP was used for sequence similarity searches against a reference sequence protein database [50]. ExPASy (https://web.expasy.org/protparam/, last accessed: 14 May 2022) was used to compute the isoelectric point and molecular weight of putative CsDGAT proteins identified herein. The subcellular localization of the 10 identified CsDGATs was analyzed with the Softberry (http://linux1.softberry.com/, last accessed: 14 May 2022) and CELLO (http://cello.life.nctu.edu.tw/, last accessed: 14 May 2022) tools [51].

Sequence Similarity and Phylogenetic Analyses
The amino acid sequences of DGATs in a range of representative eukaryotic plant species, including monocotyledons, eudicots, fern, mosses, and algae, were downloaded from the NCBI and Phytozome 12.0 database (Table S1) [29]. These DGAT sequences and the CsDGAT sequences identified herein were aligned using ClusalW and used to construct a neighbor-joining tree with 1000 bootstrap replicates using MEGA 7.0 [52].

Cis-Acting Element Analyses
The C. sativa genome was queried to identify the promoter sequences (defined as the 2 Kb region upstream of the start codon) for each of the identified CsDGAT promoter sequences, and the numbers and types of cis-acting elements in these promoters were analyzed with the Online PlantCARE software program (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/, last accessed: 15 June 2022) [57]. Results were then visualized with PowerPoint, GraphPad Prism, and TBtools [53].

Subcellular Localization Analyses
The Primer 5.0 software was used to design primers, including enzyme cleavage sites (Table S2), after which the full-length coding sequence (CDS) of each candidate gene was cloned into the pBWA(V)HS-GFP vector. The resultant pBWA(V)HS-CsDGATs-GFP plasmids were introduced into Agrobacterium tumefaciens (EHA105) via electroporation, with empty plasmids serving as a negative control. Monoclonal cells that were positive for successful transformation were cultured in liquid LB medium containing kanamycin (50 mg/L) and were collected via centrifugation, after which bacteria were suspended in a solution containing 10 mM MES (pH 5.7), 10 mM MgCl 2 , and 150 µM acetosyringone. The suspension was cultured in the dark to an OD 600nm of 0.8-1.0. Then, this inoculum was used to infiltrate the lower epidermis of 4-week-old Nicotiana benthamiana leaves using a needleless syringe until the leaves had been fully penetrated. These tobacco leaves were cultivated in the dark for 3 days, after which a confocal microscope (Nikon C2-ER) was used to visualize the GFP fluorescent signal.

Analyses of CsDGAT Expression Profiles in Different Tissues and Varieties
RNA-seq data were downloaded from the NCBI (PRJNA498707) corresponding to the female inflorescences of 9 different hemp varieties, including the Mama Thai (MT), White Cookies (WC), Canna Tsu (CT), BlackLime (BL), Temple (T), Cherry Chem (CC), BlackBerry Kush (BB), Sour Diesel (SD), and Valley Fire (VF) varieties. Meanwhile, transcriptomic data corresponding to the roots, stems, leaves, flowers, and seeds of the 'Longdama No. 9' hemp variety were generated by our team, and the RNA libraries were sequenced on the Illumina NovaseqTM 6000 platform by OE Biotech, Inc., Shanghai, China (PRJNA899681). In addition, transcriptome data from hemp seeds at different developmental periods (10d, 20d, 27d) after fertilization were used to analyze the expression profiles of CsDGAT genes at different developmental stages of hempseeds (PRJNA513221). Heat maps were constructed using TBtools. In addition, samples of the root, stem, leaf, flower, and seed tissues from female hemp plants were collected in October 2021 and snap-frozen with liquid nitrogen for subsequent qPCR analysis.

Cold Treatment and qPCR
Cold treatment and gene expression analyses were performed using the 'Longdama No. 9' hemp cultivar, breeding from the Industrial Hemp Group of the Institute of Industrial Crops (Heilongjiang Academy of Agricultural Science). All hemp seedlings were grown in a greenhouse at 25 • C under an artificial light source providing 16 h of light and 8 h of dark per day. Seedlings were grown until exhibiting four pairs of true leaves, at which time they were exposed to cold (4 • C). Leaves were collected from these plants at baseline before cold exposure (0 h) and at 12, 24, 48, and 72 h of cold exposure, with three repeat experiments (n = 3 plants/experiment) for each treatment. All samples were snap-frozen with liquid nitrogen and stored at −80 • C.
An RNA extraction kit (Omega Bio-Tek, Shanghai, China) was used to extract total RNA from these frozen samples, after which an EasyScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China) were used to prepare cDNA based on provided directions. The TransStart ® Top Green qPCR SuperMix (Transgen, Beijing, China) was then used to perform qPCR based on provided directions. Actin served as a reference gene, and the comparative 2 −∆∆CT method was used to assess relative gene expression.
Three biological replicates and three technical replicates were included in all experiments. Primers used for this analysis are provided in Supplementary Table S3.

Statistical Analysis
GraphPad Prism 8.0 software was used for one-way ANOVA, and Duncan's test was used for multiple comparisons and significance of differences analysis, where the graphical data were the mean of three or more replications.

Conclusions
In the present study, the CsDGAT gene family was herein analyzed, identifying 10 candidate CsDGAT genes classified into four subfamilies. In summary, a comprehensive and systematic bioinformatics analysis was carried out on the members of the CsDGAT family, including homologous relationships, chromosomal locations, collinearity, conserved amino acids, gene structures, conserved motifs, evolutionary relationships, and cis-acting elements associated with these CsDGAT family members, in order to understand their potential gene functions better. To further understand how they may regulate hemp growth and development, the subcellular localization and the expression patterns of these CsDGATs were also analyzed. The results showed that most of the genes have different expression patterns in different varieties, tissue parts, and seed development stages, indicating that their functions are space-time differences in their functions. In addition, the cold response pattern of the CsDGATs gene was analyzed, which showed that it had different expression patterns in leaf and root tissues. Together, these results offer a valuable foundation for further studies of the functional roles of CsDGAT genes, enabling future functional validation studies while supporting efforts to improve hempseed oil composition and cold resistance.